DGAT inhibition at the post‐absorptive phase reduces plasma FA by increasing FA oxidation

In this Correspondence, A. Sharma & C. Wolfrum report that DGAT1/2 pharmacological inhibition at post‐absorptive phase in mice leads to increased fatty acid oxidation and reduced plasma fatty acid levels, which could open new therapeutic avenues to avoid GI complications observed in clinical trials.

e) I don't see the point to include in this version of the manuscript Figure 1h.First of all, why the authors studied the expression of the genes related to lipid metabolism in the liver?They do not show any functional assays (like FAO) done in this organ.Second, in contrast to this what is stated in the manuscript, some significant changes in the expression of genes related to lipid metabolism are presented in Figure 1h (at least 3 genes).Without further molecular and functional analyses, these data are simply impossible to interpret.f) What is the purpose of using DGAT1/2 inhibitors in mice exposed to the cold?I think these experiments are irrelevant in the context of re-proposing DGAT1/2 inhibitors as a therapy against obesity.At least in the current form of the manuscript, the link is not explained.
Minor: a) In the sentence: "In humans, DGAT1 seems to be indispensable for dietary lipid absorption (...) " a supporting reference is missing.b) In the same sentence: "(...) while in mice DGAT2 efficiently compensates for the loss of DGAT1 activity (Chitraju et al., 2019) " -this might suggest compensation for intestinal DGAT1, which is misleading since Chitraju et al. describes such an overlap in the adipose tissue.c) "Consequently, although DGAT1 loss of function in humans causes severe diarrhea-like symptoms, Dgat1 (...) " -the gene name writing should be unified.d) Figure 1 g description: "Etomoxir slightly decreased the FAP (...) "-typo e) Curves on graphs in Figure 1 b-d are not described.Similarly, the bars in Figure 1 g, h.f) The authors should indicate the number of biological replicates used for each experiment.
Referee #2 (Novelty/Model system Comments for Author): The study relies solely on the use of chemical inhibitors of DGAT and activators of AMPK to draw the conclusion, which are no specific and inconclusive.More specific approaches such as DGAT1/2 KO mice and ectopic expression of constitutively active AMPK, should be used.
Referee #2 (Remarks for Author): In this short communication, the authors provided a single figure showing that pharmacological inhibition of DGAT1/2 at postoperative phase reduces plasma FFAs by increasing FAO.However, the findings are rather preliminary with little mechanistic explanation.1) Although the authors observed increased FAO by DGAT1/2 inhibitor in brown adipocytes (panel g), it does not decreased plasma FFAs in mice is attributed to increased FAO.There are many other factors which can modulate FFA levels such as lipolysis, lipogenesis.
2) There is no mechanistic explanation at all on how pharmacological inhibition of DAGT1/2 can increase FAO.
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We are thankful to both the reviewers for very insightful comments.Incorporating information to address these points will definitely improve the manuscript.We would like to highlight that we were over-conscious of exceeding the word limit which led some aspects remain incompletely discussed and explained.As you would see from our response to specific comments, majority of these points can be addressed by adding existing information and provide focused arguments in the manuscript text.We hope that with our response, the reviewers are persuaded that the data presented here are sufficient to justify as a Correspondence.
Referee #1 (Remarks for Author): In this short correspondence, Sharma and Wolfrum present a novel use of DGAT 1 and 2 inhibitors as anti-obesity agents that reduce plasma levels of free fatty acids via increasing fatty acid oxidation in the adipose tissue.The data shown are interesting and might be of clinical importance in the future especially if gastrointestinal side effects of DGAT1i are avoided when following the protocol proposed by the authors.However, several concerns should be addressed to support the claims of the authors.Honestly, I am not sure if the experiments required to support the claims of the authors can be presented in a short format of "Correspondence".However, the concept is definitely worth bringing forward.
We thank the reviewer for very positive feedback.
a) It is claimed that the observed effect of DGAT1/2 inhibition was absorption-independent but it was not really verified.Why was the timing 3 h post food withdrawal chosen as free from absorption?For this, either the activity of intestinal DGAT1/2 or lipid transport across the intestine should be determined at given conditions of drug administration.
We agree with the reviewer that this is a crucial point to be addressed.Our conclusions are based on the following points: A) The 3 h post food withdrawal was selected based on our experience with the Fat Tolerance Test (oral gavage of olive oil and monitoring the clearance).In our experience, the peak of the plasma fat reaches in 2 hours and the majority is cleared in the 4h.Since we administered the DGATi at 3h post food withdrawal, the absorption and action of DGATi coincide with the time point at which most of the gastric fat is cleared.Moreover, we have done one batch of experiments with 5h food withdrawal; overall the results were consistent but towards the later time points the fasting effect comes in, therefore we selected 3h food withdrawal which clears gastric fat but doesn't invoke a fasting response yet.
B) Chitraju et al recently characterized adipose-specific Dgat1/2 double knockout and found a similar effect i.e., reduced circulating FA combined with reduced RER.This indicates that adipose-specific loss of DGAT1/2 loss of activity is the key driver of this phenotype (Chitraju et al., 2023, eLife).In this report, authors saw an increased Ucp1 expression in the non-brown adipose depots and speculated that the browning could be a driver of this phenotype.However, our ongoing experiments suggest that Ucp1 upregulation may not be responsible for this phenotype but instead be a consequence (please see next point).Mechanistically, AMPK activation by AMP and Acyl-CoA is a much more likely possibility and we have included that in the revised manuscript.We also observed a diarrhoea-like symptom in non-fasted mice while this symptom was mitigated when the drug was administered after 3h of food withdrawal.We will add this information in the revised manuscript.E) If the phenotype was due to defective lipid absorption, one would expect a higher RER due to increased carbohydrate oxidation which is not the case here.
Moreover, DGAT1/2 will block the synthesis of triglycerides from the existing free fatty acids (monoglycerides) -so where do these fatty acids come from?
As the author rightly pointed out, the DGATs block the re-esterification.The origin of these intracellular lipid is from basal lipolysis which is always happening in the adipose tissue even in the fed state (and this constitutes a futile lipid cycle).After DGAT inhibition, the fatty acids that were to be re-esterified are now free to be used for FAO.In this regard, AMPK seems to play an important role of facilitate and sustain the FAO.We have now edited the manuscript text to clarify this issue.
b) The authors claim that their results might bring back DGAT1/2 inhibitors into clinical focus.However, in their study, they used only a single dose of the inhibitors.What happens when multiple doses of the inhibitors are used?Is the effect of these compounds still absorption-independent?As evident from the adipose-specific Dgat1/2 knockout data presented above, the results are most likely to be chronic.However, at this point, we have no data on chronic pharmacological treatment.However, we believe that this short report is more like a proof of concept that needs further exploration, especially in a pilot clinical trial.c) It is not described which type of adipose tissue is studied, both in vitro and in vivo and this will have systemic consequences -does DGAT1/2 inhibition increase fatty acid utilization in white adipose and blocks the formation of fat depots or boosts the function of brown adipose?What about FAO in other organs?In our recent manuscript (which is foundational work for this report, Sharma et al., 2023, Mol Metab) we have shown that in vitro DGATi increases FAO in both white and brown adipose tissue.A hallmark of this effect was a large increased OCR.We also performed a seahorse assay with HepG2 cells (unpublished data).We found a similar trend, but the magnitude was much smaller.This is likely because the DGAT activity is highest in the adipose tissue.We can add this data in the revised manuscript.
Effect of DGATi on the OCR in HepG2 cells (left) or brown adipocytes in vitro (right).In the previous version of the manuscript, we showed a significant drop in the RER which was coincident with the reduced plasma fatty acid levels.That suggested that in vivo, FAs are oxidized more in the presence of DGATi.With the description of the adipose-specific knockout mice, I hope that the reviewer is convinced about the specificity.We will briefly clarify this aspect in the revised manuscript.
e) I don't see the point to include in this version of the manuscript Figure 1h.First of all, why the authors studied the expression of the genes related to lipid metabolism in the liver?They do not show any functional assays (like FAO) done in this organ.Second, in contrast to this what is stated in the manuscript, some significant changes in the expression of genes related to lipid metabolism are presented in Figure 1h (at least 3 genes).Without further molecular and functional analyses, these data are simply impossible to interpret.
As suggested by the reviewer, we will remove this data point.f) What is the purpose of using DGAT1/2 inhibitors in mice exposed to the cold?I think these experiments are irrelevant in the context of re-proposing DGAT1/2 inhibitors as a therapy against obesity.At least in the current form of the manuscript, the link is not explained.The main objective for the comparison to RT vs cold was to see if FA availability is the major driver of the phenotype.Also, it is known that cold exposure also leads to a higher rate of re-esterification by DGATs, a phenomenon known as futile cycling.It was important to differentiate the effect of DGATi at resting and at cold-stimulated state.Consistently, in the cold-exposed mice, the reduction in the RER was more pronounced.We will clarify this point in the revised manuscript.
Minor: a) In the sentence: "In humans, DGAT1 seems to be indispensable for dietary lipid absorption (...) " a supporting reference is missing.b) In the same sentence: "(...) while in mice DGAT2 efficiently compensates for the loss of DGAT1 activity (Chitraju et al., 2019) " -this might suggest compensation for intestinal DGAT1, which is misleading since Chitraju et al. describes such an overlap in the adipose tissue.c) "Consequently, although DGAT1 loss of function in humans causes severe diarrhea-like symptoms, Dgat1 (...) " -the gene name writing should be unified.d) Figure 1 g description: "Etomoxir slightly decreased the FAP (...) "-typo e) Curves on graphs in Figure 1 b-d are not described.Similarly, the bars in Figure 1 g, h.f) The authors should indicate the number of biological replicates used for each experiment.We thank the reviewer for these inputs.These points will be taken care of in the revised manuscript.
The study relies solely on the use of chemical inhibitors of DGAT and activators of AMPK to draw the conclusion, which are no specific and inconclusive.More specific approaches such as DGAT1/2 KO mice and ectopic expression of constitutively active AMPK, should be used.
We thank the reviewer for the very helpful suggestion.Although we are unable to provide data on the constitutive active AMPK mouse model, we believe that our discussion on the adipose-specific Dgat1/2 double knockouts substantiates our conclusions.Moreover, we now provide another reference that very strongly suggests that AMPK could be the key driver.
Moreover, very recently, we thoroughly validated the role of AMPK signalling in mediating the role of DGATi in adipocytes (Sharma et al., 2023, Mol Metab).We will include some more information in the revised text to highlight this aspect.
Pinkosky et al. showed that AMPK can also be directly activated by activated fatty acids (Acyl-CoA).Since after the blockage of DGATs, there will be an excess pool of Acyl-CoA, this provides a link between the two pathways.We will include this information and a short schematic for a better representation of this mechanism.*Pinkosky, S.L., Scott, J.W., Desjardins, E.M. et al.Long-chain fatty acyl-CoA esters regulate metabolism via allosteric control of AMPK β1 isoforms.Nat Metab 2, 873-881 (2020).https://doi.org/10.1038/s42255-020-0245-2Referee #2 (Remarks for Author): In this short communication, the authors provided a single figure showing that pharmacological inhibition of DGAT1/2 at post-operative phase reduces plasma FFAs by increasing FAO.However, the findings are rather preliminary with little mechanistic explanation.1) Although the authors observed increased FAO by DGAT1/2 inhibitor in brown adipocytes (panel g), it does not decreased plasma FFAs in mice is attributed to increased FAO.There are many other factors which can modulate FFA levels such as lipolysis, lipogenesis.2) There is no mechanistic explanation at all on how pharmacological inhibition of DAGT1/2 can increase FAO.
We agree with the reviewer that we did not present much of the mechanistic data here.This is also partly because we recently established the role of AMPK signaling in mediating the effect of DGATi in adipocytes.We will provide a better discussion on this in the revised manuscript.
1) We agree that a reduced plasma FA could originate from different mechanisms.However, our in vivo indirect calorimetry data clearly indicates reduced RER which is because of increased FAO at the systemic level.Therefore, the two data together suggest FAO.Moreover, our better description of the phenotype of the adipose-specific Dgat1/2 knockout mice unequivocally proves the adipose-centric FAO in vivo.2) We will edit the text to better explain the mechanistic activation of AMPK by DGATi as we have published this aspect very recently.In addition, we will add a small schematic for a better representation (Fig below I have carefully read your letter and discussed it with my colleagues here.As mentioned in my initial decision letter, we agree with the referees that the concept developed in your manuscript is worth being spread to the community.Given the extent of the referees' concerns, we thought a longer article might allow you to address all the issues raised.However, from your letter I understand this might not be feasible, and we thus decided to reconsider our decision and invite revision of your manuscript as a "correspondence".As you rightly noted, we are flexible in terms of formatting, and could accommodate longer text.
Please note however that a revised version should nevertheless address the main referees' concerns and that the data should convincingly support the conclusions.The manuscript will be subjected to re-review, and I cannot guarantee at this stage that the outcome will be favorable.
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Please note: When submitting your revision you will be prompted to enter your funding and payment information.This will allow Wiley to send you a quote for the article processing charge (APC) in case of acceptance.This quote takes into account any reduction or fee waivers that you may be eligible for.Authors do not need to pay any fees before their manuscript is accepted and transferred to the publisher.EMBO Press participates in many Publish and Read agreements that allow authors to publish Open Access with reduced/no publication charges.Check your eligibility: https://authorservices.wiley.com/author-resources/Journal-Authors/openaccess/affiliation-policies-payments/index.html We would like to thank both the reviewers for their insightful comments.Addressing these points has improved the manuscript.We would like to stress that we strictly adhered to the word limit, which led to some aspects remaining incompletely discussed and explained.As you can see from our response to specific comments, these points are now addressed by adding supporting data and focused arguments/explanations in the manuscript text.We hope that the revised version is acceptable as a Correspondence.
Referee #1 (Remarks for Author): In this short correspondence, Sharma and Wolfrum present a novel use of DGAT 1 and 2 inhibitors as anti-obesity agents that reduce plasma levels of free fatty acids via increasing fatty acid oxidation in the adipose tissue.The data shown are interesting and might be of clinical importance in the future especially if gastrointestinal side effects of DGAT1i are avoided when following the protocol proposed by the authors.However, several concerns should be addressed to support the claims of the authors.Honestly, I am not sure if the experiments required to support the claims of the authors can be presented in a short format of "Correspondence".However, the concept is definitely worth bringing forward.
We thank the reviewer for very positive feedback.We agree that adding more mechanistic data would have been interesting, nevertheless, we believe that the revised manuscript is suitable and acceptable as a Correspondence.
a) It is claimed that the observed effect of DGAT1/2 inhibition was absorption-independent but it was not really verified.Why was the timing 3 h post food withdrawal chosen as free from absorption?For this, either the activity of intestinal DGAT1/2 or lipid transport across the intestine should be determined at given conditions of drug administration.
We agree with the reviewer that this is a crucial point to be addressed.We have added some data (Fig 2B,lower panel) and few other circumstantial evidence to prove the specificity of the phenotype.
A) If the phenotype was due to compromised lipid absorption, one would expect a higher RER due to increased carbohydrate oxidation which is not the case here.Consistently, when we performed a refeeding experiment after D1+2i administration, we observed and increased RER, thus demonstrating the reduction in RER is not related to absorption and reduction in the plasma FAs is most likely a consequence of FAO.
B) The 3 h post food withdrawal was selected based on our experience with the Fat Tolerance Test (oral gavage of olive oil and monitoring the clearance).In our experience, the peak of the plasma fat reaches in 2 hours and the majority is cleared in the 4h.Since we administered the DGATi at 3h post food withdrawal, the absorption and action of DGATi coincide with the time point at which most of the intestinal fat is cleared.Moreover, we have done one batch of experiments with 5h food withdrawal; overall the results were consistent but towards the later time points the fasting effect comes in, therefore we selected 3h food withdrawal which clears gastric fat but doesn't invoke a fasting response yet.C) Chitraju et al recently characterized adipose-specific Dgat1/2 double knockout and found a similar effect i.e., reduced circulating FA combined with reduced RER.This indicates that adipose-specific loss of DGAT1/2 loss of activity is the major driver of this phenotype (Chitraju et al., 2023, eLife).
D) The acute and visually discernible gastrointestinal side-effect of combined DGATi in HFD-fed mice (and a corresponding effect of DGAT1i in humans) is watery diarrhoea.We observed a diarrhoea-like symptom in non-fasted mice while this symptom was mitigated when the drug was administered after 3h of food withdrawal.
27th Jul 2023 1st Authors' Response to Reviewers Moreover, DGAT1/2 will block the synthesis of triglycerides from the existing free fatty acids (monoglycerides) -so where do these fatty acids come from?
As the reviewer rightly pointed out, DGATs block the re-esterification.The origin of these intracellular lipid is from basal lipolysis which is always happening in the adipose tissue even in the fed state (and this constitutes a futile lipid cycle).After DGAT inhibition, the fatty acids that were to be re-esterified are now free to be used for FAO.In this regard, AMPK seems to play an important role of facilitate and sustain the FAO.We have now edited the manuscript text to clarify this issue.Unfortunately, we cannot cite many of these excellent studies due to limited number of citations allowed in a Correspondence.b) The authors claim that their results might bring back DGAT1/2 inhibitors into clinical focus.However, in their study, they used only a single dose of the inhibitors.What happens when multiple doses of the inhibitors are used?Is the effect of these compounds still absorption-independent?
As evident from the adipose-specific Dgat1/2 double knockout data discussed above, the results are most likely to be chronic.However, at this point, we have no data on chronic pharmacological treatment.In one set of experiment, we observed a three-day consecutive injection was still consistent.We believe that this short report is more like a proof of concept that needs further exploration, especially in a pilot clinical trial.
c) It is not described which type of adipose tissue is studied, both in vitro and in vivo and this will have systemic consequences -does DGAT1/2 inhibition increase fatty acid utilization in white adipose and blocks the formation of fat depots or boosts the function of brown adipose?What about FAO in other organs?In our recent manuscript (which is foundational work for this report, Sharma et al., 2023, Mol Metab) we have shown that in vitro DGATi increases FAO in both white and brown adipose tissue.A hallmark of this effect was a large increased OCR.We also performed a seahorse assay with HepG2 cells.We found a similar trend, but the magnitude was much smaller.Therefore, adipose tissue seems primary effector also because the rate of basal lipolysis and DGAT activity is highest in the adipose tissue.We have added these data in the revised manuscript with appropriate discussion (Fig 2D).
d) Although the impact of DGAT1/2 inhibition on fatty acid metabolism in adipocytes in vitro is clear, such a link in vivo is not proven.The authors should be either more careful when postulating the impact of DGAT1/2 blockage on FAO specifically in adipose or determine FAO in DGAT1/2 adipocyte-specific knockout animals upon the treatment with inhibitors.
We agree that other organs may also be involved in FAO oxidation.We have modified our statement to make this distinction as suggested.
We would like to highlight a recent study by Chitraju et al., (eLife, 2023) where adipose-specific knockout of Dgat1/2 led to reduced RER, indicating that the adipose tissue may be the major effector of this phenotype.
e) I don't see the point to include in this version of the manuscript Figure 1h.First of all, why the authors studied the expression of the genes related to lipid metabolism in the liver?They do not show any functional assays (like FAO) done in this organ.Second, in contrast to this what is stated in the manuscript, some significant changes in the expression of genes related to lipid metabolism are presented in Figure 1h (at least 3 genes).Without further molecular and functional analyses, these data are simply impossible to interpret.
Thanks, you for this suggestion.We have now removed these data.
f) What is the purpose of using DGAT1/2 inhibitors in mice exposed to the cold?I think these experiments are irrelevant in the context of re-proposing DGAT1/2 inhibitors as a therapy against obesity.At least in the current form of the manuscript, the link is not explained.
The main objective for the comparison to RT vs cold was to see if FA availability is the major driver of the phenotype.It is known that cold exposure also leads to a higher rate of lipolysis and also reesterification by DGATs, a phenomenon known as futile cycling.It was important to differentiate the effect of DGATi at resting and at cold-stimulated state.Consistently, in the cold-exposed mice, the reduction in the RER was more pronounced.We have clarified this point in the revised manuscript.We thank the reviewer for these inputs.These points are taken care of in the revised manuscript.
Referee #2 (Novelty/Model system Comments for Author): The study relies solely on the use of chemical inhibitors of DGAT and activators of AMPK to draw the conclusion, which are no specific and inconclusive.More specific approaches such as DGAT1/2 KO mice and ectopic expression of constitutively active AMPK, should be used.
We thank the reviewer for his/her helpful suggestions.Although we are unable to provide data on the constitutive active AMPK mouse model, we believe that our discussion on the adipose-specific Dgat1/2 double knockouts substantiates our conclusions.Moreover, we now provide another reference that very strongly suggests that AMPK could be the key driver.
Recently, we thoroughly validated the role of AMPK in mediating the role of DGATi in adipocytes (Sharma et al., 2023, Mol Metab).We will include some more information in the revised text to highlight this aspect.Similarly, Pinkosky et al. showed that AMPK is directly activated by Acyl-CoA.Since D1+2i will lead to excess Acyl-CoA, activation of AMPK by AMP as well as by Acyl-CoA is the mechanistic aspect that links the two pathways.We have included a schematic for a better representation of this mechanism (Fig 2E Referee #2 (Remarks for Author): In this short communication, the authors provided a single figure showing that pharmacological inhibition of DGAT1/2 at post-operative phase reduces plasma FFAs by increasing FAO.However, the findings are rather preliminary with little mechanistic explanation.1) Although the authors observed increased FAO by DGAT1/2 inhibitor in brown adipocytes (panel g), it does not decreased plasma FFAs in mice is attributed to increased FAO.There are many other factors which can modulate FFA levels such as lipolysis, lipogenesis.
2) There is no mechanistic explanation at all on how pharmacological inhibition of DAGT1/2 can increase FAO.
We agree with the reviewer that we did not present much of the mechanistic data here.This is also partly because we recently established the role of AMPK signaling in mediating the effect of DGATi in adipocytes.We now provide a better discussion on this in the revised manuscript.Thank you for the submission of your revised manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you in this busy time of the year.We have now received the reports from the 2 referees who re-reviewed your manuscript.As you will see, they still raise the following concerns on your work: 1/ single dosage of DGAT inhibitor 2/ lack of mechanistic insight However, they also mention that "the possible medical impact is high" (referee #1) and that "[the study] might be acceptable as a short communication.However, the limitation of this study should be discussed."(referee #2).Having discussed your manuscript a second time with my colleagues, and taking into account the practical limitations discussed previously, we agree that the article is worth being published as a correspondence with the results at hand.However, we would like you to clearly state the limitations of the work in your manuscript, and overstatements should be avoided.
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3/ Please provide a complete author checklist, which you can download from our author guidelines (https://www.embopress.org/page/journal/17574684/authorguide#submissionofrevisions).Please insert information in the checklist that is also reflected in the manuscript.The completed author checklist will also be part of the RPF.4/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329),EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the RPF.Additional experiments are required to justify the claims raised in the manuscript.At the current stage, the manuscript is not bringing sufficient advancements over previously published papers: https://doi.org/10.1016/j.molmet.2023.101701and https://doi.org/10.7554/eLife.88049.2However, the possible medical impact is high, and generally, the methodology is adequate.
Referee #1 (Remarks for Author): The authors have replied to most of the issues raised by the reviewer and overall, the manuscript is stronger in that form.Discussing point-by-point the limitations of the study balances the authors' claims and increases manuscript credibility.Also, including the data from re-feeding + DGAT1/2 inhibition supports the idea that applying the inhibitor 3 h after food withdrawal limits the contribution of intestinal DGATs.Still, that reasoning is deductive and indirect, while the statement could gain solid support by simply showing directly a minimized DGAT1/2 activity.That is also reflected in the nomenclature used, eg.'intuitively', and 'plausibly', when speaking about the data which should be solid.The authors also claim that the presented data are kind of "proof of concept that needs further exploration, especially in a pilot clinical trial".At this stage, this is an overstatement.This claim would be justified if the authors would provide a piece of evidence that multiple doses (weeks of dosing) of an inhibitor are safe to use at least in rodents.Otherwise presented data are not bringing forward the concept of usage of DGAT inhibitors beyond cited by the authors publications: https://doi.org/10.1016/j.molmet.2023.101701and https://doi.org/10.7554/eLife.88049.2 Minor: a) it should be clearly indicated in the main text that a single dose of inhibitors was tested in the study b) the information about sex and age of animals are missing c) in Figure 2 B please change x numeration to a 24 h clock format Referee #2 (Novelty/Model system Comments for Author): The conclusion was based solely on the use of a single dosage of DGAT inhibitor.

Referee #2 (Remarks for Author):
In this revised version, the authors did not provide any additional data to address my two major concerns: (1) decreased plasma FFAs in mice is attributed to increased FAO; (2)There is no mechanistic explanation at all on how pharmacological inhibition of DAGT1/2 can increase FAO.Instead, they provided further explanation to support their conclusion.This might be acceptable as a short communication.However, the limitation of this study should be discussed.

Response to reviewers
Referee #1 (Novelty/Model system Comments for Author): Addi onal experiments are required to jus fy the claims raised in the manuscript.At the current stage, the manuscript is not bringing sufficient advancements over previously published papers: h ps://doi.org/10.1016/j.molmet.2023.101701and h ps://doi.org/10.7554/eLife.88049.2However, the possible medical impact is high, and generally, the methodology is adequate.
Referee #1 (Remarks for Author): The authors have replied to most of the issues raised by the reviewer and overall, the manuscript is stronger in that form.Discussing point-by-point the limita ons of the study balances the authors' claims and increases manuscript credibility.Also, including the data from re-feeding + DGAT1/2 inhibi on supports the idea that applying the inhibitor 3 h a er food withdrawal limits the contribu on of intes nal DGATs.S ll, that reasoning is deduc ve and indirect, while the statement could gain solid support by simply showing directly a minimized DGAT1/2 ac vity.That is also reflected in the nomenclature used, eg.'intui vely', and 'plausibly', when speaking about the data which should be solid.The authors also claim that the presented data are kind of "proof of concept that needs further explora on, especially in a pilot clinical trial".At this stage, this is an overstatement.This claim would be jus fied if the authors would provide a piece of evidence that mul ple doses (weeks of dosing) of an inhibitor are safe to use at least in rodents.Otherwise presented data are not bringing forward the concept of usage of DGAT inhibitors beyond cited by the authors publica ons: h ps://doi.org/10.1016/j.molmet.2023.101701and h ps://doi.org/10.7554/eLife.88049.2 Minor: a) it should be clearly indicated in the main text that a single dose of inhibitors was tested in the study b) the informa on about sex and age of animals are missing c) in Figure 2 B please change x numera on to a 24 h clock format We thank the reviewer for highligh ng the relevance of our work and assessment of the methodology.We have taken care of the feasible points raised by the reviewer.We agree with the reviewer that addi onal future studies are needed to consolidate the concept described here.For clarity, we have now included a sec on in the manuscript to underline the limita ons of this study.
We would like to emphasise that we understand the seriousness of overstatements as one quoted by the reviewer ("proof of concept that needs further explora on, especially in a pilot clinical trial").We would like to men on that this statement was made in our response to reviewer's comment and not in the manuscript.In the manuscript text, as rightly pointed out by the reviewer, we have used nondefini ve terms to dis nguish findings from assump ons.
Regarding the x-axis of graphs in 2B, since the total dura on of the measurement is less than 24h, we have plo ed me on the x-axis with respect to the treatment point because it is easier to assess the dura on of the effect of inhibitor treatment.
Regarding the cited papers, we acknowledge that the cited papers (one of them is from us) made the founda on of this work.However, neither of those studies relate to pharmacological aspects of DGAT inhibitors, which is the topic of this correspondence.The conclusion was based solely on the use of a single dosage of DGAT inhibitor.
Referee #2 (Remarks for Author): In this revised version, the authors did not provide any addi onal data to address my two major concerns: (1) decreased plasma FFAs in mice is a ributed to increased FAO; (2)There is no mechanis c explana on at all on how pharmacological inhibi on of DAGT1/2 can increase FAO.Instead, they provided further explana on to support their conclusion.This might be acceptable as a short communica on.However, the limita on of this study should be discussed.
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...] The figure on the left is from Chitraju et al., (2023) where HFD fed the Dgat1/2 double knockout shows a drastic reduction in the RER.[...] D) The acute and visually discernible gastrointestinal side-effect of combined DGATi in mice (and a corresponding effect of DGAT1i in humans) is watery diarrhoea, especially if the mice are on HFD.

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For data quantification: please specify the name of the statistical test used to generate error bars and P values, the number (n) of independent experiments (specify technical or biological replicates) underlying each data point and the test used to calculate p-values in each figure legend.The figure legends should contain a basic description of n, P and the test applied.Graphs must include a description of the bars and the error bars (s.d., s.e.m.).See also 'Figure Legend' guidelines: https://www.embopress.org/page/journal/17574684/authorguide#figureformat 8 the sentence: "In humans, DGAT1 seems to be indispensable for dietary lipid absorption (...) " a supporting reference is missing.b) In the same sentence: "(...) while in mice DGAT2 efficiently compensates for the loss of DGAT1 activity (Chitraju et al., 2019) " -this might suggest compensation for intestinal DGAT1, which is misleading since Chitraju et al. describes such an overlap in the adipose tissue.c) "Consequently, although DGAT1 loss of function in humans causes severe diarrhea-like symptoms, Dgat1 (...) " -the gene name writing should be unified.d) Figure 1 g description: "Etomoxir slightly decreased the FAP (...) "-typo e) Curves on graphs in Figure 1 b-d are not described.Similarly, the bars in Figure 1 g, h.f) The authors should indicate the number of biological replicates used for each experiment.
** Reviewer's comments ***** Referee #1 (Novelty/Model system Comments for Author): Novelty/Model system Comments for Author): ). Inhibition of DGATs increases Acyl CoA levels and AMP levels that may activate and facilitate the AMPK-mediated FAO.Thank you for your appeal asking us to reconsider our decision on your manuscript.
1) We agree that a reduced plasma FA could originate from different mechanisms.However, our in vivo indirect calorimetry data clearly demonstrate a reduced RER which is because of increased FAO at the systemic level.Therefore, two data together suggest FAO.Moreover, our better description of the phenotype of the adipose-specific Dgat1/2 knockout mice unequivocally proves the adipose-centric FAO in vivo.2) We have edited the text to better explain the mechanistic activation of AMPK by D1+2i.In addition, we added a schematic for a better representation (Fig 2E).Inhibition of DGATs increases Acyl-CoA levels and AMP levels that plausibly activate and facilitate the AMPKmediated FAO.

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